One important protein in primary hemostasis is von Willebrand Factor (vWF). Plasma von Willebrand Factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, and especially the very large vWF multimers are haemostatically competent. The existence of plasma factors that control the size of vWF multimers has long been suspected. The von Willebrand Factor-cleaving protease (“vWF-cp”) is involved in the limitation of platelet thrombus growth by proteolytic cleavage of von Willebrand Factor multimers in man (Furlan et al., (1996) Blood 87: 4223-4234). Recently, the molecular structure of von Willebrand Factor-cleaving protease and the corresponding gene have been described (WO 02/42441; Zheng et al., (2001) J. Biol. Chem. 276: 41059-41063) and have been identified as a new member of the ADAMTS family and designated ADAMTS13. vWFcp regulates vWF multimer size by proteolytic cleavage.
The large and ultra large vWF multimers play a central role in arterial thrombosis, whereby unusually large mutlimers of vWF have been seen in two similar forms of thrombotic microangiopathy—thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS)—both resulting in formation of platelet aggregation leading to disseminated occlusions in the microcirculation. Patients with TTP have a deficiency of vWF-cp, whereas patients with HUS show normal activity of the protease.
There are several types of TTP: An acute idiopathic or sporadic form, an intermittent form with an eventual relapse, and a chronic relapsing form. Chronic relapsing TTP is associated with acquired or congenital deficiency of vWF-cp. The rare hereditary form of TTP has been related to specific gene mutations in the ADAMTS-13 locus. Acute idiopathic TTP or acquired TTP is usually more severe than chronic relapsing TTP, wherein these patients have acquired antibodies against vWF-cp, which inhibit the von Willebrand Factor-cleaving protease (Furlan et al., (1998) Blood 91: 2839-2846; Furlan et al., (1998) N. Engl. J. Med. 339: 1578-1584). Acquired TTP also occurs occasionally during pregnancy or in the postpartum period. Intermittent relapsing TTP is also associated with the reappearance of vWF-cp inhibitor. For other forms of TTP, such as ticlopidine-associated TTP, it has also been observed that these patients have acquired antibodies against vWF-cp (Moake, (2002) N. Eng. J. Med. 347:589-600). However, some patients with acquired TTP having unusually large vWF multimers in plasma lack severe reduced levels of vWF-cp.
In general, inhibitory antibodies against proteins cause serious problems, for example within the coagulation cascade, leading to blood loss or thrombosis.
Congenital and acquired TTP are discriminated by the presence of inhibitory antibodies against vWF-cp in the plasma of up to 80% of patients suffering from acquired TTP, and total absence of vWF-cp in plasma of patients with hereditary TTP. So far, inhibitory antibodies in plasma of patients are determined by static enzyme assays under non-physiological conditions and confirm the diagnosis of acute, antibody-mediated TTP.
Different assays of vWF-cp for diagnosis of congenital and acquired TTP have been described. vWF-cp activity and the presence of inhibitors of vWF-cp are determined by incubation of purified vWF multimers with plasma samples of patients, followed by immunoblotting of degraded vWF substrate with anti-vWF antibodies and multimer analysis (Furlan et al., (2002) Sem. Thromb. Haemost. 28:167-172). The method is very sensitive in the range of low protease activity; however, the accuracy is only moderate in the subnormal or normal range of protease activity. A collagen-binding assay for determination vWF-cp activity and vWF-cp inhibitors as described by Gerritsen et al. [(1999) Thromb. Haemost. 82:1386-1389] can be completed within 6 hours, but the method is less sensitive in the very low range of protease activity as compared to the immunoblotting of degraded vWF multimers (Furlan et al. 2002 supra). The assays described in the prior art, however, are very cumbersome, time consuming and require the expertise of laboratories familiar with the technique. Moreover, the known prior art assays only allow for detection of vWF-cp inhibitors that impair the catalytic function of vWF-cp. Inhibitory antibodies which may impair a vWF-cp function other than the catalytic activity, e.g. endothelial cell binding, cannot be detected by these assays.
Therefore a need exists for a test system that allows the detection and determination of anti-vWF-cp antibodies in a patient's plasma that impair vWF-cp function other than the enzyme's catalytic protease activity.